2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-<i>endo</i> ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-<i>O</i>-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification

Additional file 3: of The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

Mammalian two-hybrid analysis of Aes-mediated Gro/Tle repression. 20 ng of the expression constructs (pMCamVP16, pMCTle4VP16 or pMCTle1VP16) were co-transfected with 70 ng of firefly luciferase reporter construct pLucF24ZF, 20 ng of the expression construct for the six-domain of human Six3 (pMChSix3(85-203)mZFb6) and full length Aes (pKCAes at the indicated concentrations) into HeLa cells. The interaction of the artificial leucine zipper domain “acid” and “base” [63] was used as a control. All three interactions were set 100% in the absence of Aes (acid/base corresponds to a more than 100 fold activation of the luc reporter construct compared to a control without bait, similar induction rates were seen for Tle4/Six3 and Tle1/Six3). Addition of the Aes expression construct at the indicated concentrations did not affect the acid/base interaction (blue bars), but strongly reduced the Tle4/Six3- (red bars) and the Tle1/Six3 interaction (green bars). (PDF 10028 kb

Additional file 4: of The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

MO-induced gro/tle loss of function phenotypes. Embryos at the 1-cell stage were co-injected with morpholino oligonucleotides and 1 μg/ml FITC-dextran. Injections were performed using either a single morpholino directed against tle1 (C), tle2b (D,F), and tle3b (E), or combinations directed against tle1 + 2b (H), tle1 + 3b (I), tle2b + 3b (J), and tle1 + 2b + 3b (K). Single morpholino oligonucleotides were injected at a concentration of 600 μM (C-F) and combinatorial injections (H-K) were performed using 300 μM of each MO. Phenotypes of FITC-dextran positive embryos were observed after the beginning of eye pigmentation at stage 32 (B-F) and stage 28 (G-K). (B,G) Wild type control embryos were injected with 1× Yamamoto’s and FITC-dextran. All embryos are shown in dorsal view with anterior at the top. Compared to the wild type controls (B,G), morpholino injected embryos developed smaller eyes that were shifted towards the midline (D,E,J,K) or cyclopic eyes (C,H,I). In rare cases the eyes were lost entirely (E). (A) Phenotypes of FITC-positive embryos were categorized at stage 28-32 into weak and strong phenotypes. Weak phenotypes developed smaller eyes that were shifted towards the midline, whereas strong phenotypes showed cyclopic eyes. Eye-less phenotypes were included into the group of strong phenotypes. Abbreviations: MO, morpholino oligonucleotide; WT, wild type. Scale bar 100 μM. (PDF 10028 kb

Additional file 2: of The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

Gro/Tle dependence of 1 tcf3 in gain-of-function experiments. Embryos at the 1-2 cell stage were co-injected with 40 ng/μl of the indicated gfp:HSE:Tcf3 constructs. Heat treatment (10 min, 43.5 °C) was applied at stage 14. (A) Statistical overview of the phenotype distribution. Whole mount in situ hybridization experiments for rx2 were performed on embryos at stage 21. (B) dorsal view of a stage 31 embryo with anterior at the top, (C) lateral view with anterior at the left. Arrowheads indicate ectopic otic vesicles, the arrow points to the endogenous otic vesicle. Scale bar 100 μm; B and C. (PDF 10028 kb